(I-339) Generation of Fat-Cartilage Microphysiological Model as a New Tool to Study Obesity-Associated Osteoarthritis

Afflicting over 500 million patients worldwide, osteoarthritis (OA) is a physically and mentally debilitating disease that alters joint pathology [1]. Despite societal prevalence, direct causation is unknown, and there is no disease modifying osteoarthritis drugs (DMOADs) reaching FDA approval.

One of the main risk factors of OA is obesity, and with the current obesity epidemic, there is interest in how these two comorbidities relate [2]. It has been recognized that obesity is a systemic disease impacted by adipose tissue malfunction. Namely, pro-inflammatory adipokines are secreted from obese adipose tissue into the bloodstream and nearby tissues, resulting in global chronic, low-grade inflammation [3]. The correlation between obesity and OA was originally perceived as due to increased mechanical loading; however, the pro-inflammatory adipokines secreted from hypertrophic adipose tissues are also seen in osteoarthritic joints, suggesting a biochemical correlation [3-5]. Therefore, there is an experimental need to study how obese adipose tissues impact the joint microenvironment to increase the risk of OA.

To overcome the limitations of current in vitro and animal models in simulating OA, our lab has recently developed a multicomponent microphysiological system (MPS) that utilizes human bone mesenchymal stem cells (hBMSCs) to create joint tissues (miniJoint) [6]. Herein, we report modification of the miniJoint to establish a fat-cartilage MPS that focuses on the direct effect of obese-like adipose tissues on cartilage. The objective is to utilize this modified microphysiological model to define the biochemical correlation between obesity and OA and to test for potential therapeutics.

Differentiate hBMSCs into adipose and cartilage within an MPS system: The process of creating fat-cartilage MPS was modified from the previous methods in generating miniJoint [6]. Briefly, hBMSCs (P5) were encapsulated in 15% methacrylated gelatin (GelMA) and photocrosslinked within resin inserts. Inserts were placed within dual-flow bioreactors supplemented with differentiation mediums (Figure 1). 6 sets of adipogenic and 6 sets of cartilage bioreactors were established. After a 28-day differentiation process, Luminex and ELISA were employed to analyze tissue phenotypes.

Assemble fat-cartilage MPS: After differentiation, the bottom flows of an adipose and cartilage bioreactor were connected to establish a fat-cartilage MPS (Figure 2). 3 fat-cartilage MPSs were established. In the bottom flow of each MPS, a common medium simulating “synovial fluid” was pushed from the adipose bioreactor to the cartilage to allow for tissue crosstalk. 1 of the MPSs was supplemented with adipogenic medium, while the other 2 contained adipogenic medium with sodium palmitate to induce obese-like changes in fat [7, 8].

Test potential drug in fa-cartilage MPS: After 28 days, Urolithin A (UA), a natural metabolite previously shown as a promising therapeutic agent in OA treatment [9, 10], was supplemented to the chondrogenic, adipogenic, and synovial-like fluids of one of the obese fat-cartilage MPSs for 7 days to test its potential in treating obesity-associated osteoarthritss. The medium will not be changed for the other MPSs. All groups were then collected and analyzed.

Following analysis via real-time quantitative PCR (RT-qPCR), histology, and Luminex assays, each fat-cartilage MPS group displayed distinct phenotypes based on treatment.

Comparing the adipose tissue (AD) of the groups treated with sodium palmitate to the AD of the control group, AD exposed to sodium palmitate displayed obese-like changes. Staining of the sodium palmitate-treated AD revealed evidence of hypertrophy and hyperplasia compared to control AD, which correlate to the development of obese-like phenotype. RT-qPCR analysis revealed a significant upregulation of leptin and downregulation of adiponectin, an important feature of adipose tissues in obese individuals. There were also distinct markers of adipose dysfunction, including increased adipsin, PPARγ, and TNF-α expression and decreased adipsin expression.

Cartilage exposed to this obese-like tissue showed changes like those seen in OA, such as an increase in articular cartilage degradation markers (MMP13, ADAMTS4, and ADAMTS5) and pro-inflammatory cytokines (IL-6, IL-8, and TNF-α). We saw a decrease in aggrecan, indicating an unhealthy cartilage extracellular matrix.

Additionally, treating AD with experimental therapeutics, Urolithin A, partially prevented the presentation of an obese-like phenotype, displaying levels more like those without sodium palmitate exposure. This then limited the detrimental effects of obese AD on the cartilage tissue unit, lowering the presentation of OA markers seen in the group solely treated with sodium palmitate.

With the successful formation of an adipose-cartilage model, we can confirm the biochemical correlations between obesity and osteoarthritis. Specifically, we can find the biochemical pathways mediating this correlation, elucidating potential pathways to alleviate symptoms of obesity-associated OA. In the future, the bi-directionality between the obese adipose and the cartilage can be established to see whether this causes further adipose and cartilage dysfunctions. Additionally, the creation of an obese adipose-cartilage MPS creates a robust tool to screen drugs to treat these comorbidities, as seen with the treatment of Urolithin A. The potential to target adipose specifically to reverse an osteoarthritic phenotype within cartilage can be a revolutionary tool in combating obesity-associated OA.

This work was carried out with the support and resources at the Lin Laboratory within the Department of Orthopaedic Surgery at the University of Pittsburgh. This work was funded by the University of Pittsburgh Swanson School of Engineering Summer Undergraduate Research Internship and the Department of Orthopaedic Surgery at the University of Pittsburgh. 

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